Field of the Invention
The present invention relates to the field of nanodevices and nanosensors for single cell injection, patterning and detection of biomolecules.
Related Art
Presented below is background information on certain aspects of the present invention as they may relate to technical features referred to in the detailed description, but not necessarily described in detail. That is, individual parts or methods used in the present invention may be described in greater detail in the materials discussed below, which materials may provide further guidance to those skilled in the art for making or using certain aspects of the present invention as claimed. The discussion below should not be construed as an admission as to the relevance of the information to any claims herein or the prior art effect of the material described.
Nanopipette technology has been shown to be a capable platform for many applications. Actis et al., (including two of the present inventors) developed a sensing platform, named STING (“Signal Transduction by Ion Nano Gating”), where the very tip of a quartz nanopipette is functionalized with chemical or biological receptors (Actis, P., O. Jejelowo, and N. Pourmand, Ultrasensitive mycotoxin detection by STING sensors. Biosensors & Bioelectronics, 2010. 26(2): p. 333-337). The nanometer scale opening at the tip creates a region that is sensitive to analyte binding with the attached receptors. Additionally, Rodolfa, et al., have shown that nanopipettes can be used for controlled deposition onto functionalized surfaces (Rodolfa, K. T., et al., Two-component graded deposition of biomolecules with a double-barreled nanopipette. Angewandte Chemie-International Edition, 2005. 44(42): p. 6854-6859). Further work has demonstrated deposition of material onto a surface in an inorganic solvent (Rodolfa, K. T., et al., Nanoscale pipetting for controlled chemistry in small arrayed water droplets using a double-barrel pipet. Nano Letters, 2006. 6(2): p. 6). Individual molecules were delivered to cells' plasma membranes using nanopipettes (Bruckbauer, A., et al., Nanopipette delivery of individual molecules to cellular compartments for single-molecule fluorescence tracking. Biophysical Journal, 2007. 93: p. 3120-3131).
Laforge et al. developed an electrochemical attosyringe, based on a nanopipette that delivers liquid by applying a voltage across the liquid/liquid interface formed at the nanopipette opening (Laforge, F. O., et al., Electrochemical attosyringe. Proceedings of the National Academy of Sciences of the United States of America, 2007. 104(29): p. 11895-11900). The resulting force is sufficiently strong to induce the flow of liquid into/out of the pipette. They have successfully used this effect to deliver femtoliters of aqueous solution into mammalian cells in culture. Carbon nanopipettes have shown efficacy in cell injection as well. Pressure driven injection of fluorescent dyes into oral squamous carcinoma cells was demonstrated by Schrlau, et. al. (Carbon nanopipettes for cell probes and intracellular injection. Nanotechnology, 2008: p. 015101-1-4).
Cell Patterning
Controlling cell attachment and growth has emerged as an important topic in biological disciplines from neuroscience to stem cell research (James, C. D., et al., Aligned microcontact printing of micrometer-scale poly-L-lysine structures for controlled growth of cultured neurons on planar microelectrode arrays. Ieee Transactions on Biomedical Engineering, 2000. 47(1): p. 17-21; Welle, A., et al., Photo-chemically patterned polymer surfaces for controlled PC-12 adhesion and neurite guidance. Journal of Neuroscience Methods, 2005. 142(2): p. 243-250). By controlling where and how cells grow and mature, specific characteristics can be induced during cell growth. For instance, it has been shown by Oliva, et. al. (Patterning axonal guidance molecules using a novel strategy for microcontact printing. Neurochemical Research, 2003. 28(11): p. 1639-1648), that neuron differentiation can be controlled by chemical cues, allowing the direction of axon growth to be predetermined and customized for specific experiments. Similarly, inkjet printing has been shown to be capable of controlling neural stem cell differentiation (Ilkhanizadeh, S., et al., Inkjet printing of macromolecules on hydrogels to steer neural stem cell differentiation. Biomaterials, 2007. 28(27): p. 3936-3943).
Cell patterning has been investigated using a multitude of methods. By leveraging the advancements made in semiconductor fabrication technology patterns with feature sizes small enough to guide single cells has been made possible. The most widely used cell patterning technique is microcontact printing (μCP), whereby a master mold is fabricated using traditional photolithography, and an elastomeric stamp is created from the master. The stamp can then be inked with biomolecules and the pattern can be applied to an arbitrary substrate (Wilbur, J. L., et al., Microfabrication by microcontact printing of self-assembled monolayers. Advanced Materials, 1994. 6(7-8): p. 600-604). This method has seen wide success in controlling cell growth, particularly in the arena of controlled neural growth (Park, T. H. and M. L. Shuler, Integration of cell culture and microfabrication technology. Biotechnology Progress, 2003. 19(2): p. 243-253). Photolithography itself can be used for patterning as well. By exposing a substrate to radiation through a mask, surface chemistry can be modified, thus allowing specific attachment molecules to be placed in the appropriate regions (Welle, A., et al., Photo-chemically patterned polymer surfaces for controlled PC-12 adhesion and neurite guidance. Journal of Neuroscience Methods, 2005. 142(2): p. 243-250). Topographic cues have been shown to control cell growth as well. By changing the surface roughness that cells interface with cells adhesion can be controlled. The combination of chemical and topographical attachment cues has even been demonstrated as leading to improved cell differentiation (for example, Stenger, D. A., et al., Microlithographic determination of axonal/dendritic polarity in cultured hippocampal neurons. Journal of Neuroscience Methods, 1998. 82(2): p. 167-173). The drawback to these methods is that once the pattern has been designed and fabricated it cannot be changed without designing a new mask and restarting the entire process from scratch. Some methods have been developed for patterning that do not rely on standard semiconductor fabrication technology, and therefore are not limited by the fabrication process. Gustaysson, et al., (Neurite guidance on protein micropatterns generated by a piezoelectric microdispenser. Biomaterials, 2007. 28(6): p. 1141-1151) demonstrated a piezo actuated microdispenser capable of depositing 100 μL droplets with a precision of 6-8 μm. Dip pen and fountain pen lithography allows spot sizes as small as 40 nm to be deposited in an arbitrary user defined pattern as shown by Schmidt, R. C. and K. E. Healy (Controlling biological interfaces on the nanometer length scale) in Journal of Biomedical Materials Research Part A, 2009. 90A(4): p. 1252-1261. However this technique is limited to deposition of only a single pattern at a given time.
By using quartz nanopipette technology described below, the pattern deposited on a substrate is computer controlled and can thus be modified at any time by the user, and is capable of easily depositing multiple patterns that are registered relative to each other. The pattern can be used to accomplish cell attachment, as shown in FIG. 2, to a substrate 216, where a cell 212 is attached to a point on the substrate where an adhesion material has been deposited.
Cell Injection
Methods for cell injection have historically used a pulled glass micropipette. Traditional micropipettes suffer from several drawbacks including large size relative to typical cells, low cell viability, lack of feedback and the requirement of a skilled operator (Pillarisetti, A., et al., Evaluating the effect of force feedback in cell injection. Ieee Transactions on Automation Science and Engineering, 2007. 4(3): p. 322-331; Stephens, D. J. and R. Pepperkok, The many ways to cross the plasma membrane. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(8): p. 4295-4298).
A multitude of other methods for cell injection have been developed to alleviate these drawbacks. Methods such as electroporation, and the use of the pore forming toxin streptolysin-O (SL-O) have been developed for passive transfer of material into the cell (Wang, M. Y., et al., Single-cell electroporation. Analytical and Bioanalytical Chemistry, 2010. 397(8): p. 3235-3248; Knight, D. E. and M. C. Scrutton, Gaining access to the cytosol—the technique and some applications of electropermeabilization. Biochemical Journal, 1986. 234(3): p. 497-506; Giles, R. V., et al., Selecting optimal oligonucleotide composition for maximal antisense effect following streptolysin O-mediated delivery into human leukaemia cells. Nucleic Acids Research, 1998. 26(7): p. 1567-1575). Electroporation has been demonstrated to induce transient permeability in the cell membrane by application of high voltage, after which material can diffuse in. Cells have shown the ability to heal SL-O lesions under certain circumstances. In both cases, stress is introduced to the cell, and in the case SL-O lesions uptake is limited to ˜100 kDa.
Direct methods of cell injection have been demonstrated using other unrelated nanofabricated structures. A nanoneedle, fabricated on AFM tips and coated with DNA was inserted into a single cell. Injection was accomplished by diffusion of DNA from the nanoneedle (Sung-Woong, H., et al., High-efficiency DNA injection into a single human mesenchymal stem cell using a nanoneedle and atomic force microscopy. Nanomedicine: Nanotechnology, Biology and Medicine, 2008: p. 215-25). Similarly, quantum dots were delivered by cleavage of disulfide bonds linking the dots to a nanoneedle within a cell (Chen, X., et al., A cell nanoinjector based on carbon nanotubes. Proceedings of the National Academy of Sciences of the United States of America, 2007. 104(20): p. 8218-8222).
Single Cell In Vivo Detection of Intracellular Molecular Species
Several methods allowing intracellular recording have been developed. Kopelman and coworkers (Tan, W., et al., Submicrometer intracellular chemical optical fiber sensors. Science 258, 778-781, doi:10.1126/science.1439785, 1992; Barker, S. L. R., et al., Cellular Applications of a Sensitive and Selective Fiber-Optic Nitric Oxide Biosensor Based on a Dye-Labeled Heme Domain of Soluble Guanylate Cyclase. Analytical Chemistry 71, 2071-2075, doi:10.1021/ac9901081, 1999) pioneered the application of chemical modified tapered optical fiber for the extracellular monitoring of pH and nitric oxide. Vo-Dihn and coworkers reported the analytical application of antibody modified optical fiber for measurement of a fluorescent analyte in a single cell (Vo-Dinh, T., et al., Antibody-based nanoprobe for measurement of a fluorescent analyte in a single cell. Nat Biotech 18: 764-767, 2000). One of the advantages of employing tapered optical fiber relies on high spatial resolution achievable using near field scanning optical microscopy. Sensors must be carefully manipulated under a microscope to avoid damage to cells. In addition to these physical constraints, the selective detection of biomolecules by affinity methods is itself challenging due to the many interfering species inside the cytoplasm. More recently an optical fiber nanobiosensor was constructed to detect a cancer biomarker into a single cell through an enzymatic sandwich immunoassay (Zheng, X. T. & Li, C. M. Single living cell detection of telomerase over-expression for cancer detection by an optical fiber nanobiosensor. Biosensors and Bioelectronics 25: 1548-1552, 2010). Electrical detection methods are known to be more suitable than other methods due to improved durability, sensitivity, rapid response, and integration with other device components. Even so, electrical-based sensors for intracellular measurements face many challenges. Microelectrodes, are usually large enough to damage typical mammalian cells (5 to 10 μm), and procedures are often limited to measurements in oocytes and embryos, which are at least ten times larger. Recently, microelectrodes protruding inside cells were employed to measure sub-threshold synaptic potentials by Hai, A., et al. (In-cell recordings by extracellular microelectrodes.) in Nature Methods (2010) 7, 200-202. Despite successful intracellular cation and pH sensors using microelectrodes coated with ion-selective membranes by Bakker, E. & Pretsch, E. (Nanoscale potentiometry.) in TrAC Trends in Analytical Chemistry 27, 612-618, the intracellular electrical detection of biomolecules remains elusive. Lieber et collaborators developed a novel approach where a nanoscale field effect transistor (nanoFET) modified with phospholipid bilayers was able penetrate a single cell and record intracellular potentials (Tian, B. et al. Three-Dimensional, Flexible Nanoscale Field-Effect Transistors as Localized Bioprobes. Science 329, 830-834, doi:10.1126/science.1192033, 2010). However no electrical sensors were employed to detect biomolecular interaction inside a single cell.
STING (“Signal transduction by ion nano-gating”) technology has also been shown capable of detecting DNA, proteins and mycotoxins in a sample (Fu, Y., et al., Nanopore DNA sensors based on dendrimer-modified nanopipettes. Chem Commun (Camb), (2009), 4877-4879, doi:10.1039/b910511e; Umehara, S., et al., Label free biosensing with functionalized nanopipette probes. Proceedings of the National Academy of Sciences (2009) 106, 4611-4616, doi:10.1073/pnas.0900306106; Actis, P., et al., Ultrasensitive mycotoxin detection by STING sensors. Biosensors and Bioelectronics (2010)26, 333-337). Based on a functionalized quartz nanopipette, STING technology does not require any nanofabrication facility; each probe can be easily and inexpensively tailored at the bench. Receptor molecules can be incorporated using well established surface chemistries. Besides biosensing, nanopipette platform were used to study single molecule biophysics, controlled delivery inside individual cells, and to image cells at the nanoscale (Clarke, R. W., et al., Trapping of proteins under physiological conditions in a nanopipette. Angew Chem Int Ed Engl (2005), 44, 3747-3750, doi:10.1002/anie.200500196; Laforge, F. O., et al., Electrochemical attosyringe. Proceedings of the National Academy of Sciences (2007), 104, 11895-11900, doi:10.1073/pnas.0705102104; Klenerman, D. & Korchev, Y. Potential biomedical applications of the scanned nanopipette. Nanomedicine (Lond) (2006), 1, 107-114, doi:10.2217/17435889.1.1.107). Vitol and coworkers developed a SERS active carbon nanopipette for intracellular analysis (Singhal, R. et al. Small diameter carbon nanopipettes. Nanotechnology, (2010), 015304). SERS functionality was added by incorporating gold nanoparticles on the outer surface pipette tip. SERS spectra obtained with the nanopipette inserted within the nucleus show typical features associated with DNA.
However, there remains a need in the art for a nanopipette biosensor that can operate within a living cell.